T. Mikita et Gp. Beardsley, EFFECTS OF ARABINOSYLCYTOSINE-SUBSTITUTED DNA ON DNA RNA HYBRID STABILITY AND TRANSCRIPTION BY T7 RNA-POLYMERASE/, Biochemistry, 33(31), 1994, pp. 9195-9208
Cytosine arabinoside (araC) is a potent antileukemic agent which inter
feres with DNA replication both as a dNTP competitive inhibitor as wel
l as after its misincorporation into DNA. We previously developed a ch
emical methodology for the synthesis of DNA oligomers containing araC
which allowed us to study its site specific effects on duplex stabilit
y and chemical reactivity [Beardsley, G. P., Mikita, T., Klaus, M., an
d Nussbaum, A. (1988) Nucleic Acids Res. 16, 9165], as well as its eff
ects on DNA ligase and DNA polymerase activity [Mikita, T., and Beards
ley, G. P. (1988) Biochemistry 27, 4698]. The DNA polymerase studies,
in addition to other observations, showed that araC in DNA templates c
ould have an inhibitory effect on polymerase bypass. As a template les
ion, there exists the potential for interference with other aspects of
DNA metabolism, such as transcription. We have characterized a DNA/RN
A hybrid containing an araC-G base pair, comparing thermal stability,
chemical cleavage rates, and duplex gel mobility to an identically seq
uenced DNA duplex. We find that the A-form DNA/RNA hybrid and the B-fo
rm DNA duplex are nearly identical in the extent their thermal stabili
ty is affected by an araC-G(dG) base pair. Substitutions of araC for d
C were made at various positions in a series of DNA duplex substrates
containing a T7 RNA polymerase promoter with variable length coding st
rands. These were used to probe the effect of araC on promoter recogni
tion, initiation, and elongation by T7 RNA polymerase in vitro. Substi
tutions in the central promoter region had no observable effect on RNA
polymerase binding, initiation rate, or transcriptional output. Codin
g strand substitutions defined an area of high sensitivity in the init
iation region where miss-starts, primer slippage, and an inability to
escape from abortive cycling occur depending on the position substitut
ed. Substitutions after position 10 had little effect on transcription
output. These highly variable, position dependent effects indicate a
narrow window of vulnerability where transcription output is severely
reduced (similar to 100-fold) by a subtle DNA lesion that has little o
r no consequence when situated elsewhere in these small coding units.